br Material and methods br Results br Discussion Organisms

Material and methods
Results
Discussion Organisms have developed a variety of anti-glycation defense mechanisms. In Gramine australia to the aldose-reductase-pathway or 2-oxoaldehyde dehydrogenase-pathway, the glyoxalase system is the only mechanism, which is not restricted to liver [19]. This system converts methylglyoxal into D-lactate and thus it detoxifies this cytotoxic compound, which is also one of the most potent precursors of AGE formation. Consequently, a decrease in the activity of this detoxification pathway may accelerate the process of AGE formation and accumulation during aging. Therefore, the aim of this study was to examine the cell type and age-dependent localization and activity of the enzyme glyoxalase I in the human brain. The study was expected to suggest a possible connection between the enzymatic activity of glyoxalase I in old age and the previously described age-dependent deposition of AGEs in the Brodmann area 22 [10]. The age-dependency of glyoxalase I expression shows in interesting biphasic response. The number of glyoxalase I immunopositive neurons and astroglia increases up to approximately 55 years of age, and decreases progressively thereafter. This peculiar time course of expression was confirmed by biochemical investigations whereby the expression of the glyoxalase I protein determined by Western blotting and its enzymatic activity was highest in brains of middle-aged donors and reduced in old age. The decreasing glyoxalase I levels strongly correlate with accumulating levels of AGEs in the same temporal region (Brodmann area 22) which we have previously investigated [10]. A similar age-dependent pattern of glyoxalase I expression has also been also shown for experimental animals. For example, the specific activities of glyoxalase I, determined in liver, spleen and kidney, increased in mice up to 12–14 months depending on the type of the tissue, thereafter decreased progressively in the old animals [15]. In aging rats, activities of glyoxalase I is also markedly decreased with age (at 30 months) in liver, whereas the activity of glyoxalase I in muscle was maintained at control level [7]. Interestingly, such a biphasic change in glyoxalase I activity has also been described during the maturation and aging of a defined cell type, the human erythrocyte. In these cells, glyoxalase I activity increased markedly during maturation of reticulocytes to erythrocytes, followed by a decrease in glyoxalase I activity from the mature to old red blood cell fraction [11]. The age-dependent changes in glyoxalase I expression could be transcriptionally and/or translationally regulated. For this reason we also measured the age-dependent RNA levels of glyoxalase I by RT-PCR. The RNA levels showed also a biphasic course very similar to those observed in protein determination. Since expression of glyoxalase I is regulated by responsive elements for insulin, Zn2+ and NF-κB [13], the initial increase in transcription activity may contribute to increased glyoxalase I RNA and finally protein levels from adulthood to middle age. NF-κB activity is reported to be elevated under conditions of oxidative stress [20], and increase of glyoxalase I transcription and translation may reflect a compensatory mechanism to deal with increased radical and carbonyl levels. All investigated brain tissues were obtained after relatively long post mortem times (Table 2) which in terms may affect enzymatic activities. But in all three groups the mean post mortem times (Table 2) are very similar and thus the measured glyoxalase I activities should be comparable among each other. However, the rate-limiting step in glyoxalase I activity is the reaction of the dicarbonyl compound with reduced glutathione. In our assay, glutathione was added in excess to measure the true activity of the enzyme. However, in the aging human brain, a decrease in the concentration of reduced glutathione caused by oxidative stress or by deficiencies in glyoxalase II activity may lead to a further decrease of its enzymatic activity in vivo [2]. Low glyoxalase I activity in old age may thus lead to a higher steady state level of reactive dicarbonyl compounds in vivo and thus contribute to the accumulation of AGEs with age in cells and tissues.